Assays for dna methylation changes

ABSTRACT

A new technique to determine the extent of DNA methylation entails generating DNA fragments of a test sample by cleaving at methylation sites that are not methylated while sparing methylation sites in the DNA that are methylated. This approach provides enhanced sensitivity to differences of even a single methylated cytosine, within or outside of a CpG island, and yet it can be employed to ascertain the methylation status of a region of DNA comprising a CpG island, a DNA region comprising one or more CpG-containing islands, or even a large variety of DNA regions.

FIELD OF THE INVENTION

The present invention relates to the detection of a modified nucleotidesequence and, more specifically, to the detection of nucleic acid basemethylation of a nucleotide sequence.

BACKGROUND OF THE INVENTION

The methylation of DNA is thought to have important effects in theregulation of gene expression in eukaryotes, having epigenetic andmutagenic effects on various cellular activities such as differentialgene expression, cell differentiation, chromatin inactivation, genomicimprinting, and carcinogenesis. Gonzalgo & Jones, Mutat. Res.386:107-118 (1997). DNA methylation is also known to have a role inregulating gene expression during cellular development. Huang et al.,Human Molecular Genetics 8: 459-470 (1999). In mammals, DNA methylationusually occurs at cytosines located 5′ of guanines, known as CpGdinucleotides. Many CpGs are clustered at/near regulatory regions ofgenes, which are called CpG islands. DNA methylation or hypermethylationwithin CpG islands is thought to be especially important in regulatinggene expression. A significant number of genes are associated with CpGislands and CpG island hypermethylation has been observed in more than90 gene promoters. CpG island hypermethylation is associated with theepigenetic inactivation of tumor suppressor or growth regulatory genesin cancer.

Because methylation of DNA often is reflective of disease status,accurate, sensitive, and efficient methods of detecting changes inmethylation patterns would be highly desirable. The existing methods ofdetecting changes in methylation patterns frequently require laborioussteps, such as subtractive hybridization and analysis using Southernblots. Existing methods that depend on reacting unmethylated cytosineswith chemical reagents like sodium bisulfite can damage templates andalter base composition, causing problems with a subsequent PCRamplification. Existing methods have other weaknesses such as aninability to detect methylation changes for the entire genome with ahigh degree of sensitivity. Accordingly, improved methods with higherthroughput and increased accuracy are needed for detecting methylationof DNA.

SUMMARY OF THE INVENTION

The present invention provides methods for detecting whether the extentof methylation of one or more regions of DNA in a test sample isdifferent from that of a control.

In one aspect, the invention comprises generating DNA fragments from atleast one test sample of DNA by cleaving methylation sites that are notmethylated while sparing methylation sites in the DNA that aremethylated, ligating an oligonucleotide-linker to the ends of the DNAfragments, amplifying the DNA by initiating amplification from thelinker, hybridizing the amplified DNA to one or more polynucleotidesimmobilized on a solid support, the polynucleotides complementary to oneor more regions of DNA in the test sample, and comparing the amplifiedDNA from the test sample that hybridizes to the immobilizedpolynucleotides versus that of a control. In an exemplary embodiment theDNA fragments may be labeled with a detectable moiety.

In another aspect, the invention comprises generating DNA fragments fromat least one test sample of DNA by cleaving methylation sites that arenot methylated while sparing methylation sites in the DNA that aremethylated, separating the fragments of appropriate sizes, ligating anoligonucleotide-linker that is labeled with a detectable moiety to theends of the DNA fragments, hybridizing the amplified DNA to one or morepolynucleotides immobilized on a solid support, the polynucleotidescomplementary to one or more regions of DNA in the test sample, andcomparing the amount of the detectable moiety associated with thepolynucleotide for the test sample versus a control.

In another aspect, the present invention uses the methods describedabove to determine if an individual has a state of disease associatedwith an abnormal extent of methylation of one or more regions of DNA inthe individual. The above aspects of the invention have many differentembodiments which will be addressed in detail below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic illustration of one embodiment of the inventivemethodology. Region A and B represent segments of genomic DNA present inDNA from a test sample and a control sample. Vertical lines indicate thelocation of methylation sites in the DNA. Vertical lines topped with an“M” indicate that the methylation site is methylated. Thus, as depicted,both DNA regions are not methylated in the control sample while region Bis methylated in the test sample. Selective amplification of regionsbordered by non-methylated methylation sites is achieved by digestingthe DNA into fragments with a methylation sensitive restriction enzyme,ligating the fragments with an oligonucleotide linker, and amplifyingthe linker ligated fragments with a linker primer. The amplified productfrom the test and control samples are differentially labeled as shownwith Cy3 and Cy5 cyanine dye. Labeled amplicons from the two samples arecombined and hybridized to an array with fixed probes for region A andB. The microarray is scanned at the excitation wavelength of 532 nm and635 nm. Dual signals for probe A indicate that region A in the testsample is not methylated while a single signal for probe B indicatesthat region B in the test sample is methylated.

FIG. 2 illustrates the use of unphosphorylated linkers in a scheme foramplifying fragments generated by cleavage with a methylation sensitiverestriction enzyme. Vertical bars represent matched base pairs. Panel Ashows a double stranded HpaII (C′CGG) linker with a top strand (SEQ IDNO: 1) and a short bottom strand with no 5′-phosphate (SEQ ID NO: 2).Panel B shows a DNA fragment following HpaII restriction. Panel C showsthe DNA formed following ligation of the unphosphorylated linker to theHpaII fragment. As seen, the short bottom strand of the linker lacking a5′ phosphate (SEQ ID NO: 2) is not ligated, leaving a nick, while thetop strand is ligated to the 5′ phosphate of the fragment (resulting inSEQ ID NO: 3). Addition of amplification reactants (polymerase anddNTPs) to the ligated fragments followed by a short incubation at 72° C.causes the un-ligated lower strand of the linker to be replaced by anewly synthesized region (see bold font) (SEQ ID NO: 4). Subsequenttemperature cycling leads to amplification by priming with unusedunphosphorylated linker top strand.

FIG. 3 illustrates the principle of amplicon preparation forinterrogating rare methylation sites by cleaving with both a methylationsensitive (“MS”) and a methylation insensitive (“MIS”) restrictionenzyme. As amplification is desired only for fragments involving an MSrestriction (MS/MS and MS/MIS). Panel A indicates that two types oflinkers are used: a phosphorylated linker for MS cleavage sites and anunphosphorylated linker for MIS cleavage sites. Panel B shows thatfollowing restriction with an MS and MIS enzyme, the lower strand of theMIS linker cannot be ligated because of the missing 5′ phosphate. Thus,a nick is present between the short bottom strand of the linker and thefragment. This non-covalently associated lower strand of the linker DNAis unstable and cannot support primer directed amplification. Thus,MIS/MIS fragments with non-covalently attached linker DNA at both endsof the fragments are not amplifiable while fragments generated by MSonly (MS/MS) or by MS at one end and MIS at the other end (MS/MIS) canbe amplified.

FIG. 4 illustrates how a linker primer can be designed to preventamplification of linker dimers. Vertical bars represent matched basepairs. Panel A shows a double stranded (SEQ ID NO: 1 and 5) HpaII(C′CGG) linker with a 5′-phosphate. Panel B shows a DNA fragmentfollowing HpaII enzyme restriction. Panel C shows a linker primer (SEQID NO: 6) for avoiding amplification of linker dimers. Panel D1 depictsa linker dimer (SEQ ID NO:7) formed from the linker in panel A. Panel D2shows a mismatched base (guanine) at the 3′ end of the linker primer(SEQ ID NO: 6) when hybridized to the bottom strand of the linker dimer(SEQ ID NO:7). The presence of a mismatched 3′ end nucleotide prohibitsthe primer from directing amplification. Panel E1 shows linker dimerligated to the HpaII fragment (resulting in SEQ ID NO: 8 and 9). PanelE2 shows that there is no mismatched base at the 3′ end of the linkerprimer (SEQ ID NO:6) when hybridized to the bottom strand of the ligatedfragment (SEQ ID NO:9). The absence of a mismatched 3′ base in thelinker primer provides for primer directed amplification. Thus,amplification of linker dimers is avoided by extending the linker primerat its 3′ end until a 3′ base incompatibility is obtained between theprimer and the primer dimer (in this example only a one base extensionis required) while maintaining 3′ end compatibility between the linkerprimer and the DNA fragment.

FIG. 5 demonstrates that the inventive methylation detection method canidentify strain specific methylation of a transgene. Panel A is aSouthern blot of mouse DNA from transgenic strains D and B hybridized toa labeled probe for the transgene. The blot shows that the transgene ismethylated in strain B but not in strain D (the increased molecularweight of the methylated (“meth”) transgene versus the unmethylated(“um”) transgene results from preparing the Southern using a methylationsensitive restriction enzyme). Panel B shows that when amplicons areprepared from strain D and B as described in Example 1, amplifiedtransgene can be detected only from strain D (the strain that does notmethylate the transgene). Gel shown panel B was prepared byelectrophoresis of transgene specific PCR of the amplicons. Arrowindicates the position of amplified transgene. Panel C shows the amountsof transgene and an unmethylated control gene present in ampliconsprepared from strain D and B as described in panel B. The amplicons fromstrain D were labeled with Cy5 while the amplicons from strain B werelabeled with Cy3. The two amplicons were combined and hybridized to anoligonucleotide microarray containing an oligonucleotide for thetransgene and one for an unmethylated control gene (both in triplicate).The table shows the amount of Cy3 and Cy5 dye hybridized to thetransgene oligonucleotide and the control gene oligonucleotide. Theobserved reduction in relative Cy3 signal indicates that the transgenein strain B (labeled with Cy3) is methylated as compared to that instrain D. In contrast, the relative similarity in Cy3 and Cy5 signal forthe control gene indicates that this gene is similarly methylated in thetwo strains of mice.

DETAILED DESCRIPTION OF THE INVENTION

As noted, methylation of DNA in mammals occurs at cytosines, includingcytosines in CpG sites, by addition of a methyl group at the pyrimidinering of the cytosine. A new approach developed herein for determiningthe extent of methylation change of one or more regions of DNA isprovided. The inventive methodology can be used to determine whether theextent of methylation of a DNA region in one sample differs from that ofa control.

In one aspect, the approach of the present invention provides anenhanced sensitivity to differences of even a single methylatedcytosine, within or outside of a CpG island, and yet it can be employedto ascertain the methylation status of a region of DNA comprising one ormore CpG islands, one or more regions of DNA comprising one or more CpGcontaining islands, or even a large variety of DNA regions. The lattermay entail determining the methylation status of between about 10,000 to50,000 or more DNA segments of the genome of a single individual. Forexample, one could use the method to interrogate the extent ofmethylation present at all known CpG rich sites (about 45,000 presentlyknown). In another aspect, hundreds or more samples can be analyzedeveryday in a standard molecular biology laboratory and a microarrayfacility. Thus, the present invention allows for rapid and relativelyinexpensive interrogation of the methylation status, genome-wide, formany individuals.

In one aspect, the present invention comprises generating DNA fragmentsfrom a sample of DNA by cleaving methylation sites in the DNA that arenot methylated while sparing methylation sites in the DNA that aremethylated. Oligonucleotide-linkers are ligated to the ends of the DNAfragments. In some embodiments, the linkers are labeled with adetectable moiety. In other embodiments, the DNA is amplified byinitiating amplification from the oligonucleotide-linkers. The DNAfragments are analyzed by hybridization to one or more polynucleotidesimmobilized on a solid support. Finally, the amount of DNA associatedwith the solid support for the test sample DNA is compared to that of acontrol to determine whether the extent of methylation of the regions ofDNA sought to be evaluated is different between the test sample and thecontrol. In some embodiments, an actual control sample of DNA is usedand the test and DNA fragments may be mixed together beforehybridization to the immobilized polynucleotides. A discussion of thevarious steps of these methods as well as additional embodiments basedon modifications and/or additions to the procedure follows.

DNA Fragmentation

DNA fragments can be generated from DNA purified from a cell, tissue orbody fluid. Techniques for isolating DNA from such sources are wellknown to those skilled in the art. See, for example, Sambrook et al.,MOLECULAR CLONING: A LABORATORY MANUAL, Vols. 1-3 (Cold Spring HarborLaboratory Press, 1989). Highly pure DNA is easy to prepare and ispreferred, but lower degrees of purity may be used provided the DNA canbe cleaved and processed as required in subsequent steps.

DNA fragments are generated by cleavage at methylation sites that arenot methylated while sparing methylation sites in the DNA that aremethylated. In this description, the phrase “methylation sites” refersto sites in the DNA that potentially could be methylated in a cell. Suchsites contain a cytosine, which is found often (but not exclusively) ina CpG context. A preferred manner for cleaving DNA fragments atmethylation sites that are not methylated, while sparing sites in theDNA that are methylated, is to cleave the DNA with one or moremethylation-sensitive agents. A “methylation-sensitive” agent is onethat normally cleaves DNA at a methylation site when the site isunmethylated, but does not cleave the site, or cleaves it at lowefficiency, when the site is methylated. A methylation-sensitive agentpreferably recognizes and cleaves only at methylation sites in the DNAwhen they are not methylated. Methylation sensitive agents thatrecognize methylation sites involving methylation of adenine or cytosinenucleotides in bacterial DNA or cytosine nucleotides in CpG, CpNpG andCCWGG sites in plant or mammal DNA can be used.

A preferred methylation sensitive agent is a methylation sensitiverestriction enzyme. Such enzymes and details for their use arecommercially available from, for example, New England BioLabs, ProMegaBiochem and Boehringer-Mannheim, among others. Techniques foridentifying other methylation-sensitive restriction enzymes are known tothe skilled artisan. Sambrook et al., supra, (1989) provides a generaldescription of methods for using restriction enzymes and other enzymes,for instance.

Illustrative methylation-sensitive restriction enzymes, suitable for usein the present invention for analyzing methylation changes in CpG sites,include HpaII (C′CGG), AciI (C′CGC), HinP1I (G′CGC), HpyCH4IV (A′CGT),EagI (C′GGCCG), NgoMIV (G′CCGGC), KasI (G′GCGCC), SmaI (CCC′GGG), BstUI(CG′CG), BspDI (AT′CGTA), BstBI (TT′CGAA), SalI (G′TCGAG), and XhoI(C′TCGAG). In accordance with the present invention, cleavage may beachieved by more than one agent, used separately or in combination.Enzymes such as EagI, NgoMIV, KasI and SmaI are especially usefulbecause they are located mainly in CpG islands and are relatively rarein other areas of the genome and the amplicons generated are of lowercomplexity, meaning that the hybridization signals will be stronger andmore specific than in the case where enzymes with a higher frequency ofrestriction sites are used. Enzymes such as HpaII (C′CGG), AciI (C′CGC),HinP1I (G′CGC), and HpyCH4IV (A′CGT) may be especially convenientbecause they produce the same “sticky” ends, allowing subsequent linkageof the cleaved end of the DNA fragment to a singleoligonucleotide-linker (i.e., a universal linker). A universal linkerwhich can be ligated to all cleavage ends generated in a particular stepof the method is preferred.

DNA fragments may be generated, at methylation sites that arenon-methylated, by using a chemical agent other than a restrictionenzyme. For example, DNA can be treated with an agent that specificallyprotects methylated DNA sites from cleavage (e.g., a methylation bindingprotein), and then the treated DNA is cleaved via an agent that cleavesthe unmethylated methylation sites. Such an agent may be amethylation-insensitive restriction enzyme or a methyl-DNA bindingprotein that can be fused to or coupled with an endonuclease which canthen specifically cut at the methylated CpG sites.

A “methylation insensitive agent” in this context is one that cleavesDNA at a cleavage site, regardless of whether the site is methylated ornot. In a preferred embodiment, the methylation insensitive agentemployed is a methylation-insensitive restriction enzyme. Exemplary ofmethylation-insensitive enzymes for use in the present invention areEcoRI (G′AATTC), ApoI (R′AATTY), Tsp509I (AATT), MseI (TTAA), BfaI(C′TAG), Csp6I (G′TAC), NIaIII (CATG′), DpnII (′GATC), CviJI (RG′CY),Sau3A (′GATC), RsaI (GT′AC), Tsp509I (′AATT), MaeI (C′TAG), NlAIII(CATG′), and DpnI (GA′TC). Other methylation insensitive agents may bechemicals, such as formic acid, hydrochloric acid, and performic acid.For example, an engineered agent such as a fusion protein comprising amethylation-specific DNA-binding protein and an endonuclease may be usedfor cleaving DNA at methylated and non-methylated methylation sites.

Ligating to Oligonucleotide-Linkers and Amplification

After generating DNA fragments by cleaving at non-methylated sites inthe DNA, the ends of the fragments are ligated to anoligonucleotide-linker. An oligonucleotide-linker may be double- orsingle-stranded and generally is from about 12 to about 24 nucleotidesin length, although shorter or longer oligonucleotide-linkers may beused. Double stranded oligonucleotide linkers are preferred. Adouble-stranded oligonucleotide-linker may be blunt-ended or have stickyends. A blunt-ended oligonucleotide-linker may be linked to ablunt-ended DNA fragment. Preferable oligonucleotide-linkers of theinvention have a sticky end that is complementary to a sticky end of aDNA fragment for which ligation is desired. Oligonucleotide-linkers maybe prepared synthetically and, if double-stranded, may be designed witha sticky end by synthesizing top and bottom DNA stands of differentlength. In another approach, the linker may be synthetically preparedwith a blunt end and then treated with an agent to create theappropriate sticky end. For example, a blunt endedoligonucleotide-linker may be treated with the same methylationsensitive restriction enzyme used to cleave the test or control DNA. Inthis case, a sticky end that is complementary will be generated on boththe DNA fragment and the oligonucleotide-linker to be ligated to thefragment.

An exemplary double stranded oligonucleotide-linker for ligation to aHpaII-digested DNA fragment can be formed by the combination of SEQ IDNO:1 and SEQ ID NO: 2, shown below (the linker is shown at top of FIG.2). This oligonucleotide linker is partially double stranded and notphosphorylated. An alternative oligonucleotide linker can be made bycombining SEQ ID NO:1 with SEQ ID NO: 5 shown below (the linker is shownat top of FIG. 4). This oligonucleotide linker is fully double strandedexcept for a two base segment reflecting the HapII site. This secondoligonucleotide linker also is phosphorylated at the 5′ end of thebottom strand as shown (see 5′ end of SEQ ID NO:5).5′-CTGCTGACGATGAGTCCTGAGT-3′ (SEQ ID NO:1; top strand) 5′-CGACTCAGGA-3′(SEQ ID NO:2; bottom strand) 5′-pCGACTCAGGACTCATCGTCAGCAG-3′ (SEQ IDNO:5; bottom strand)With a phosphorylated double stranded oligonucleotide linker such as thecombination of SEQ ID NO:1 and 5, both strands of the linker are ligatedto the cut DNA (see FIG. 4, panel E1). With a non-phosphorylated doublestranded oligonucleotide linker such as the combination of SEQ ID NO:1and 2, only one strand of the linker is ligated to the cut DNA (e.g.,the bottom strand as seen in FIG. 2, panel C). The other strand is heldin place by Watson-Crick base pairing and may be removed by denaturingconditions.

The characteristics of the oligonucleotide-linker depend on theparticular embodiment. For example, the oligonucleotide-linker may belabeled with a detectable moiety so that the DNA fragments may bedetected in later steps of the method. Labeling may be accomplished, forexample, by conjugating the detectable moiety directly to nucleotides inthe linker or the fragment, or by designing the linker to have a uniquesite for labeling, or by post amplification random labeling. A uniquesite in the linker may be a chemical linker or a capture sequence ofnucleotides. As used herein, a “capture sequence” of nucleotides is asequence designed to anneal by base pairing with a targetoligonucleotide sequence. For example, a capture sequence can be anoligonucleotide with an abiotic and GC-rich sequence that stronglybase-pairs with a complementary sequence on another nucleic acid such asa dendrimer. An “abiotic” sequence is one which is not present in thegenome of the test DNA. A dendrimer is a branched molecule to whichdozens of dye molecules can be attached (Stears et al., Physiol Genomics3: 93-99, 2000). A capture sequence can be designed into theoligonucleotide-linker to support labeling with a detectable moiety suchas a labeled dendrimer, which is described in detail below. Apost-amplification random labeling can be achieved using normal andfluorescent deoxynucleotides, a random primer, and a DNA polymerase asis well known in the art.

An oligonucleotide-linker also may be designed to provide a target forinitiating synthesis of DNA during DNA amplification. Hence, afterligation to the linker, the ends of the DNA fragments can have a uniquesequence which is of sufficient length to provide a unique recognitionsite for primers selected for in vitro DNA amplification, for example,by the polymerase chain reaction (PCR). Preferably, theoligonucleotide-linker contains a unique sequence sufficient fortargeting by the amplification primer. The unique sequence may include aportion of the linker or may include all of the linker. Anoligonucleotide linker may thus function as the linker primer. Theamplification primer also may include some of the sequence of theadjoining DNA fragment.

The length of the amplification primers for use in the present inventiondepends on several factors including the nucleotide sequence and thetemperature at which these nucleic acids are hybridized or used duringin vitro nucleic acid amplification. The considerations necessary todetermine a preferred length for an amplification primer are well knownto the person of ordinary skill in the art. For example, the length of ashort nucleic acid or oligonucleotide can relate to its hybridizationspecificity or selectivity. Because the digested fragments of DNA maycontain a complex mixture of nucleic acids, primers which are shorterthan about 12 nucleotides may hybridize to more than the linker, andaccordingly would not have sufficient hybridization selectivity foramplifying only the DNA fragments cut with a methylation-sensitiveagent. However, a 12- to 15-nucleotide sequence generally is representedonly once in a mammalian genome. Sambrook et al. (1989), supra, at pages11.7-11.8. Accordingly, to eliminate amplification of DNA fragments thatare not ligated to oligonucleotide-linkers, amplification primers arechosen which are generally at least about 14 or 15 nucleotides long.Preferably, the primers are at least about 16-17 nucleotides long, andmay even be 20-25 nucleotides long or longer. (Sambrook et al., pp.11.7-11.8). An example of an amplification primer for a HaplIoligonucleotide linker is the following: 5′-CTGACGATGAGTCCTGAGTC-3′ (SEQID NO. 6, linker primer)This primer is suitable for hybridizing and priming at theoligonucleotide-linker shown above (e.g., the double stranded linkerformed using SEQ ID NOs: 1 and 5).

Ligation of oligonucleotide-linkers to DNA is accomplished using methodswell known in the art. For example, see Sambrook et al. (1989), supra,at 1.53. This typically involves annealing of the oligonucleotide-linkerto the DNA fragment, by gradual cooling from 50° C. to 25° C., followedby ligation using 400 U of T4 DNA ligase at 16° C. The step of gradualcooling can be eliminated if blunt-ended ligation is contemplated.

By ligating an oligonucleotide-linker to both ends of a DNA fragmentgenerated by cleavage with a methylation sensitive agent, the ligatedDNA fragment will be replicated from both ends when such ends aretargeted by the amplification primer, resulting in efficientamplification, for example, by PCR (FIG. 1). Since only relatively shortfragments (e.g., those less than 1000 base pairs) can be amplified undercertain experimental conditions, the amplified fragments representregions that are not methylated. Conversely, when a region of the DNA ismethylated or has no CpG dinucleotides, it will not be cleaved by amethylation-sensitive agent, no oligonucleotide-linker will be ligated,and no amplification will result (e.g., FIG. 1). Therefore, the relativeamount of amplified fragments from specific region of test and controlsamples will reflect their relative methylation level difference.

The polymerase chain reaction is a preferred method for DNAamplification. PCR synthesis of DNA fragments occurs by repeated cyclesof heat denaturation of DNA fragments (i.e., heating to at least about95° C.), incubation at a temperature permitting hybridization of theamplification primers to all or part of the primer adapter ends of themethylation-sensitive cut DNA fragment, and primer extension. Thesecycles can be performed manually or, preferably, automatically. Thermalcyclers such as the Perkin-Elmer Cetus® cycler (Perkin-Elmer Corp.,Boston, Mass.) are specifically designed for automating the PCR processand are preferred. The number of cycles per round of synthesis generallyvaries from 2 to more than 50, the optimum number being readilydetermined by considering the source and amount of the nucleic acidtemplate, the desired yield, and the procedure to be used for detectionof the DNA. Heat stable amplification enzymes such as pwo, Thermusaquaticus or Thermococcus litoralis DNA polymerases are commerciallyavailable and eliminate the need to add enzyme after each denaturationcycle. Basic PCR techniques are described in U.S. patents by Saiki etal., see also 1988 Science 239: 487, and by U.S. Pat. No. 4,683,195, No.4,683,202 and No. 4,800,159, and other PCR variations well-known to thefield.

The precise conditions for PCR and hybridization have a much reducedeffect on the outcome of present invention as compared to that in otherPCR-based assays in cases where both amplification and hybridizationreactions are competitive: the DNA from the test and control sample iscombined and amplified simultaneously, thus, competing equally foramplification resources like the polymerase and nucleotidetriphosphates. Therefore, more accurate assays are attainable with thepresent invention, and more variation or error in performing the assayis tolerated without adversely affecting the results. Nevertheless, thevariables generally affecting PCR including temperature, salt and cationconcentration, pH, and related conditions may be optimized for eachsituation.

A variety of enzymes suitable for DNA amplification such as in PCR arewell known in the art and include, for example, Pwo, Escherichia coliDNA polymerase I, Klenow fragment of E. coli DNA polymerase I, T4 DNApolymerase, T7 DNA polymerase, Thermus aquaticus (Taq) DNA polymerase,Thermococcus litoralis DNA polymerase, SP6 RNA polymerase, T7 RNApolymerase, T3 RNA polymerase, T4 polynucleotide kinase, AvianMyeloblastosis Virus reverse transcriptase, Moloney Murine LeukemiaVirus reverse transcriptase, T4 DNA ligase, E. coli DNA ligase orQβreplicase, and the like. Mixtures of enzymes also can be used (e.g.,Pwo and Taq). Preferred amplification enzymes are the Pwo and Taqpolymerases because of their high fidelity and high processibility.Other DNA-amplification enzymes, known and yet to be discovered, may beutilized in the present invention.

It is desirable to use conditions for amplification such that the DNAfragments differing in GC content can be amplified to a similar extent.Addition of betaine (1.2 M) to a PCR reaction can be used to assist inthe amplification of GC-rich regions. Under such conditions, a region of42% GC and region of 73% GC can be amplified about equally well. Otherapproaches to achieve balanced amplification of GC rich and GC poorfragments are known in the art.

In situations where the site to be analyzed can be cleaved as a fragment(suitable for amplification) with a methylation sensitive agent, theoligonucleotide linker can be phosphorylated or unphosphorylated. Ascheme for use of a phosphorylated oligonucleotide linker is shown inFIG. 1. When a phosphorylated oligonucleotide-linker with sticky ends isused, the possibility of forming dimers of the oligonucleotide-linkerscan occur and such dimers can be ligated during the ligation step.Linker dimers may hybridize to amplification primers and be amplified,thus depleting the resources of the amplification reaction, therebyreducing the amount of amplification of ligated DNA fragments. Differentstrategies for minimizing this problem are available and include, forexample: (1) Using column chromatography, such as with asilica-gel-membrane type spin column, to purify the ligated DNAfragments from the linker dimers. In this approach, further eliminationof linker dimers may be achieved by washing the column with 30%guanidine hydrochloride prior to the regular washing step; and (2) Usinga linker primer that is comprised of a 3′-mismatched nucleotide whenhybridized to the linker dimer but a 3′ matched nucleotide whenhybridized to the linker-ligated genomic fragment. An example of thesecond approach is shown in FIG. 4.

A scheme for amplification when a unphosphorylated oligonucleotidelinker is used is shown in FIG. 2. In this embodiment, the linker isdesigned as shown with a short lower strand that is not phosphorylated.Thus, following the step of ligation, a nick or space will be presentbetween the lower strand of the linker and the fragment. Primeramplification may be achieved following ligation by a short chainextension reaction step in which polymerase and NTPs are added to thelinked fragment and the mixture incubated (e.g., 72° C.) for severalminutes. During the polymerization step, the unligated bottom strand isdenatured and removed allowing the polymerase to synthesize a bottomstrand flush to the end of the top strand. Exponential amplification maybe achieved by application of cycling temperatures. In this case, thereis no need to add a linker primer since excess top strand from theoligonucleotide linker that remains from the ligation step can serve asthe PCR primer.

In the case when a particular methylation site to be interrogated islocated too far from the next nearest methylation site, making itdifficult to amplify and thus detect a fragment of such length, the DNAcan be cleaved with both a methylation sensitive and a methylationinsensitive agent to generate amplifiable fragments. The methylationsensitive and insensitive cleavage agents may be used separately or atthe same time if compatible. A particular methylation insensitive agentcan be chosen (preferably a methylation-insensitive restriction enzyme)to provide a second cleavage site nearer to the site cleaved by themethylation-sensitive agent, yielding a fragment size more suited toamplification. Generally, fragments lengths of 50-1000 base pairs willbe suitable for amplification.

A preferred methylation insensitive agent is a methylation insensitiverestriction enzyme. The methylation insensitive restriction enzymepreferably is chosen to produce a different sticky end on the DNA thanthe end generated by the methylation-sensitive agent. In this case,oligonucleotide-linkers that are distinct from each other and whichligate only to one of the types of sticky ends generated should be used.

To avoid amplifying fragments generated solely by cleavage with themethylation insensitive agent, one may use a phosphorylatedoligonucleotide linker for ligation to the methylation sensitive siteand a unphosphorylated oligonucleotide linker for ligation to themethylation insensitive site. This approach is illustrated further inFIG. 3. As is seen in the figure, following ligation to the methylationinsensitive site, a gap or “nick” remains between one strand of thelinker and the fragment. The gap prevents amplification of the strand bya linker primer chosen to hybridize to that strand. Under suchcircumstance, exponential amplification will occur for DNA fragmentsresulting from cleavage at both ends by a methylation-sensitive agentand for DNA fragments resulting from cleavage at one end by amethylation-sensitive agent and at the other end by amethylation-insensitive agent. DNA fragments resulting from cleavage atboth ends by a methylation insensitive enzyme will not be amplified.

When both a methylation-sensitive agent and a methylation-insensitiveagent are used to cleave the DNA, oligonucleotide-linkers usedpreferably will selectively ligate only to the ends generated by one orthe other agent. Selective ligation may not be needed when each of theDNA cleavage agents are used in a separate reaction and theoligonucleotide-linker for the first reaction is added before the DNA isexposed to the second cleavage agent.

In an alternative embodiment, the step of DNA amplification may bedispensed with entirely. In this case, the ends of the DNA fragmentsgenerated by cleavage with the methylation sensitive agent are ligatedto an oligonucleotide-linker, the DNA fragments are size selected toisolate a particular size range, the fragments are labeled with adetectable moiety, and then hybridized and analyzed as described furtherahead. The DNA fragments are size selected, preferably to a size rangeof about 2,500 base pairs or less in length, more preferably about 1,500base pairs in length or less and even more preferably about 1,000 basepairs in length or less. DNA fragments may be size separated before orafter ligation to oligonucleotide-linkers. Sizing may be achieved by anymethod known in the art, including, gel electrophoresis, columnchromatography, density gradient centrifugation or ultracentrifugation(see, e.g. Steensel et al., (2001) Nature Genetics 27:304-308),filtration through membranes of controlled pore size, and the like.Filtration through membranes of controlled pore size may be achieved byapplying an external pressure or by centrifugation.

In the embodiment without amplification, the detectable moiety ispreferably attached to the oligonucleotide-linker and the moiety ispreferably of suitably high specific activity. A labeled dendrimer or afluorescent resonant energy transfer dye are examples of high specificactivity detectable moieties. The oligonucleotide linker can have acapture sequence designed for binding to a detectable moiety such as alabeled dendrimer. The oligonucleotide-linker may be labeled before orafter it is ligated to the DNA fragments.

The amount of DNA digested may be increased to improve detectionsensitivity in embodiments that do not use amplification. DNA in therange of 0.5-1.5 mg may be suitable for this purpose, although personsof ordinary skill in the art realize that the quantity of DNA sufficientto allow omitting the DNA amplification step depends on other featuresof the assay and can be readily determined without resort to undueexperimentation.

In yet another embodiment, the step of DNA amplification and the step ofligating the DNA fragments to an oligonucleotide-linker may be dispensedby using a sufficiently large amount of the DNA, size-fractionating thedigested fragments, optionally labeling the fragments through randompriming (e.g., with Cy3 and Cy5 dye-labeled dCTP), and hybridizing themto a solid support such as a microarray. For example, see Steensel etal., Nature Genet. 27: 304 (2001).

Labeling DNA

DNA can be labeled with a detectable moiety to improve detection inlater steps. Labeling can occur at any of a variety of steps in themethod. For example, the detectable moiety may be attached to theoligonucleotide-linker, which is then ligated to the DNA fragment. Inanother approach, the detectable moiety may be attached to theamplification primer, which is incorporated into the amplified DNA. Inyet another approach, the DNA fragment may be directly labeled with thedetectable moiety. In some situations, it may be preferable to labelwith a detectable moiety at more than one step (e.g., use a labeledoligonucleotide-linker and direct labeling). This may be useful whenincreased sensitivity is desired. Various combinations of detectablemoieties also may be used to label the same molecule to increase itsspecific activity.

The phrase “detectable moiety” is used here to denote any molecule (orcombinations of molecules) that may be attached or otherwise associatedwith a molecule so that the molecule can be detected indirectly bydetecting the detectable moiety. A detectable moiety can be aradioisotope (e.g., iodine, indium, sulfur, hydrogen etc.) a dye orfluorophor (e.g., cyanine, fluorescein, rhodamine), protein (e.g.,avidin, antibody), enzyme (peroxidase, phosphatase, etc.), or any otheragent that can be detected directly or indirectly. An enzyme is anexample of a detectable moiety detected by indirect means. In this case,the enzyme is attached to the target nucleic acid and the presence ofthe enzyme is detected by adding an appropriate substrate that whenacted upon by the enzyme, causes the substrate to change in color or torelease a cleavage product that provides a different color from theoriginal substrate.

A fluorescent detectable moiety can be stimulated by a laser with theemitted light captured by a detector. The detector can be acharge-coupled device (CCD) or a confocal microscope, which records itsintensity. In the case of an array, the intensities provided by thearray image can be quantified by measuring the average or integratedintensities of the spots. Interpreting the data from a microarrayexperiment can be assisted using special software, such as Dapple; see(http://www.cs.washington.edu/homes/jbuhler/research/dapple/).

A preferred detectable moiety is a cyanine dye such as Cy-5 and Cy-3.For example, test DNA fragments can be labeled with Cy-5, while controlDNA fragments can be labeled with Cy-3 (or vice versa). In this way, thetwo DNA fragment preparations may be mixed and then hybridized togetherto the solid support, scanned with the above devices at thecorresponding wavelength for each dye, with the measured densitiesproviding the ability to distinguish the relative amounts of each DNAfragment that hybridized from the two samples.

A detectable moiety may include more than one chemical entity such as influorescent resonance energy transfer (FRET). In FRET based assays,interaction between biomolecules is measured indirectly by conjugatingone of a pair of carefully selected fluorescent dyes to each of themolecules of interest. The absorption spectrum of the acceptor mustoverlap fluorescence emission spectrum of the donor, and donor andacceptor transition dipole orientations must be approximately parallel.In FRET, when these fluorescent dyes are held in close proximity(typically 10-100 Å) due to binding of the biomolecules, a uniquefluorescence signal is developed that specifically confirms theproximity and thus the binding reaction. Resonance transfer results anoverall enhancement of the emission intensity. For instance, see Ju et.al. (1995) Proc. Nat'l Acad. Sci. (USA) 92: 4347. To achieve resonanceenergy transfer, the first fluorescent molecule (the “donor” fluor)absorbs light and transfers it through the resonance of excitedelectrons to the second fluorescent molecule (the “acceptor” fluor).

In most cases, when donor and acceptor dyes are different, FRET can bedetected by the appearance of sensitized fluorescence of the acceptor orby quenching of donor fluorescence. When the donor and acceptor are thesame, FRET can be detected by the resulting fluorescence. Donor/acceptorpairs of dyes that can be used include, for example,fluorescein/tetramethylrohdamine, IAEDANS/fluroescein, EDANS/DABCYL,fluorescein/fluorescein, BODIPY FL/BODIPY FL, and Fluorescein/ QSY 7dye. See, e.g., U.S. Pat. No. 5,945,526 to Lee et al. Many of these dyesalso are commercially available, for instance, from Molecular ProbesInc. (Eugene, Oreg.).

In another embodiment, signal amplification may be achieved usinglabeled dendrimers as the detectable moiety. A dendrimer is a bulkythree dimensional molecule that can be labeled to very high specificactivity, providing signal amplification (see, e.g., Physiol Genomics3:93-99, 2000). Fluorescently labeled dendrimers are available fromGenisphere (Montvale, N.J.). These may be chemically conjugated to theDNA fragments by methods known in the art. The labeled dendrimer alsocan be attached to the amplified DNA by designing a capture sequence inthe amplification primer (e.g., a long abionic, GC-rich nucleotidesequence), which can be targeted by a complementary sequence in thedendrimer. In the event the capture sequence in the primer createsproblems during amplification, a polymerization “stop signal” can beadded 3′ of the capture sequence (between the capture sequence and theprimer sequence) so that the capture sequence in the primer will not beamplified. The C18 spacer below is an example of a useful “stop signal.”

Persons of ordinary skill in the art will realize that this is just oneexample, and many other stop signals are available and known in the art.For example several may be obtained from Integrated DNA Technologies,Inc. (Coralville, Iowa) and other sources.Hybridization to Immobilized Polynucleotides

A solid support with polynucleotides immobilized thereon is useful forcapturing DNA fragments prepared as described above to determinemethylation status for any region of the genome. The fragments arepreferably labeled with a detectable moiety as already discussed toassist in detecting the amount of the DNA that hybridizes to theimmobilized polynucleotides.

The immobilized polynucleotides are designed to specifically hybridizeto a region of the DNA for which determination of its extent ofmethylation is desired. Thus, if one is interested in determining theextent of methylation at a single site in the genome, the solid supportmay have a polynucleotide complementary to a fragment that would arisein the method if that site were not methylated. A solid support may havemultiple different immobilized polynucleotides, each designed to annealto a different DNA fragment that may be generated in the assay dependingon whether the methylation site at one end of the fragment ismethylated. As described further below, multiple polynucleotides may beimmobilized to a single support in the form of an array. The solid phasemay have from hundreds to tens of thousands of polynucleotidesimmobilized as an array, depending on the size of the array and thetechnology used for immobilization.

Quantitation of the extent of methylation of one or more regions of DNAin a test sample versus a control sample is achieved through competitiveamplification of fragments of DNA with single pairs ofoligonucleotide-linkers (or one oligonucleotide-linker in the case ofsingle enzyme digestion) and/or competitive hybridization to the solidsupport. This may be appreciated from the following example wheregenomic DNA from the test and control are digested with at least onemethylation sensitive enzyme. If 100,000 fragments are amplified usingoligonucleotide-linkers, and 100 of the fragments are differentiallymethylated between the two samples, 99,900 fragments will be amplifiedto very similar extent in both samples after the reactions aresaturated, and the 100 fragments will be amplified differentially. Aftermixing the two amplified products (each labeled with a distinct dye),hybridizing to polynucleotides immobilized on the support (e.g., amicroarray), and scanning at the specified wavelength, 99,900 wells orspots on the support will show neutral color (i.e., similar levels ofhybridization signal to the two DNA sources) while 100 wells or spotswill show a red (Cy-5) or green (Cy-3) color (i.e., differential levelsof hybridization signal to the two DNA sources), the latter therebyindicating a difference in methylation level at those sites (e.g., CpGsites).

Using the combination of Cy3 labeled test sample and Cy5 labeled controlsample, the resulting hybridization signal strength on the arrayfollowing hybridization can be interpreted as follows:

-   Red: Means differential methylation between the test sample and    control sample. The control sample dominates, so the control sample    is less methylated than the test sample.-   Green: Means differential methylation between the test sample and    the control sample. The test sample dominates, so the test sample is    less methylated than the control sample.-   Yellow: Means about equal methylation for the test sample and the    control sample.-   No color: Means either heavy methylation in both test and control    samples or the immobilized polynucleotide sequence is not present in    the genomic DNA of the test or control.-   Color intensity: Although less predictive than color, a high    intensity indicates a low overall extent of methylation for the    region in both the test and control, while a low intensity indicates    a high overall extent of methylation for the region in both the test    and control.

A. Immobilized Polynucleotides

The sequence of polynucleotides immobilized to a support can bedetermined by resort to DNA sequence readily available in publiclyavailable sequence databases, including that gleaned from the HumanGenome Project. As used herein, a polynucleotide can be a single or adouble stranded nucleic acid and may be DNA or RNA. A polynucleotideincludes at least 2 nucleotides and may be prepared synthetically or bycleaving natural nucleic acid. The term polynucleotide encompasses theterm oligonucleotide, which refers to a shorter polynucleotide ofbetween two to about 100 nucleotides in length.

Polynucleotide sequences can be designed to hybridize to virtually anyregion of the genome where methylation is suspected. A virtual (i.e.,“computer based”) restriction digestion may be performed on humangenomic DNA sequence using the recognition sequence for themethylation-sensitive agents of interest, and the identity of sequencefor the immobilized polynucleotides determined. Immobilizedpolynucleotides can be selected to interrogate particular sequences ingenes, such as the upstream regulatory region of a gene, the proteinencoding portion of the gene, or downstream sequence associated with thegene. In such cases, the immobilized polynucleotides may be designed tohybridize to a sequence in the gene or near to the gene.

The immobilized polynucleotide may be a short segment of DNA such as anoligonucleotide or the immobilized polynucleotide can be a longerstretch of DNA such as a cDNA or portion thereof or genomic fragment ofDNA. Polynucleotides also may be chosen to interrogate intergenicregions, which are referred to herein as sequence upstream of a promoterand downstream of a poly A site. In either case, the sequence ofimmobilized polynucleotides is preferably selected to hybridize to theregion of interest, but not to other regions. This can be determinedwith the aid of computer sequence programs such as BLAST.

Polynucleotides for immobilization should be designed such that theywill not hybridize to other fragments that may be generated by theparticular embodiment of the method chosen. In the case whereamplification is used, the immobilized polynucleotides preferably havesequence complementary to a segment of DNA that have potential cleavagesites for a methylation-sensitive agent. The sites should be closeenough to each other so that the fragment can be amplified under thespecified PCR conditions if the sites are not methylated, and,therefore, cleaved. Polynucleotides for immobilization are preferablyoligonucleotides which comprise a hybridizing sequence preferablybetween about 40 to 70 nucleotides in length and more preferably betweenabout 50 to 60 nucleotides in length. As already discussed, immobilizedpolynucleotides can be longer than 100 nucleotides in length and mayeven constitute a cDNA or genomic DNA fragment thereof.

B. Solid Support

Polynucleotides immobilized to a solid support are used for detectinghybridization to the amplified and detectably-labeled target nucleicacid. The polynucleotides may be immobilized on any solid support,organic or inorganic, or a combination of any of these—in the form ofparticles, strands, precipitates, gels, sheets, tubing, spheres,containers, capillaries, pads, slices, films, plates, slides, and thelike. Typical supports are made of glass, plastic, or nylon.

With respect to implementing the present invention, a solid support forimmobilizing polynucleotides preferably is flat but may take onalternative surface configurations. For example, the solid support maycontain raised or depressed regions on which polynucleotide synthesistakes place or where polynucleotides are attached. In some embodiments,the solid support will be chosen to provide appropriate light-absorbingcharacteristics. Thus, the support may be a polymerized LangmuirBlodgett film, functionalized glass, Si, Ge, GaAs, GaP, SiO₂, SiN₄,modified silicon, or any one of a variety of gels or polymers such as(poly)tetrafluoroethylene, (poly)vinylidendifluoride, polystyrene,polycarbonate, or combinations thereof. The solid support may be a glassmicroscope slide.

The surface of the solid support can contain reactive groups, whichcould be carboxyl, amino, hydroxyl, thiol, or the like suitable forconjugating to a reactive group associated with the immobilizedpolynucleotides. Polynucleotides can be attached to the solid support bychemical or physical means such as through ionic, covalent or otherforces well known in the art. Polynucleotides also can be attached to asolid support by means of a spacer molecule, essentially as described inU.S. Pat. No. 5,556,752 to Lockhart el al. A spacer molecule typicallycomprises between 6-50 atoms in length although larger and shorterspacers are possible and includes a surface attaching portion thatattaches to the solid support.

Attachment to the support can be accomplished by carbon-carbon bonds,using supports having, for instance, (poly)trifluorochloroethylenesurfaces or, preferably, by siloxane bonds, employing glass or siliconoxide as the solid support, for example. Siloxane bonding can be formedby reacting the support with trichlorosilyl or trialkoxysilyl groups ofthe spacer. Aminoalkylsilanes and hydroxyalkylsilanes,bis(2-hydroxyethyl)-aminopropyltriethoxysilane,2-hydroxyethylaminopropyltriethoxysilane, aminopropyltriethoxysilane orhydroxypropyltriethoxysilane are useful surface attaching groups.Additionally, for use in synthesis of polynucleotides, the spacer canhave a protecting group, attached to a functional group (i.e., hydroxyl,amino or carboxylic acid) on the distal or terminal end of the spacer(opposite the solid support). After deprotection and coupling, thedistal end can be covalently bound to an oligomer.

Another solid support is a microelectronic chip as described, forexample, in U.S. Pat. No. 6,051,380 to Sosnowski et al., U.S. Pat. No.6,129,828 to Sheldon, III et al., and U.S. Pat. No. 6,017,696 to Heller.This type of chip exploits electronically accelerated hybridizationconducted under very low salt conditions, which avoids problems, withDNA conformation and secondary structure, associated with otherhybridization methods. Immobilized polynucleotides can be movedelectronically to specific sites on the microchip, and thenhybridization is determined. In the electronic mediated approach, use ofan electronically mediated, active hybridization process to move andconcentrate target DNA molecules reduces the time to detecthybridization from hours (as in conventional, passive methodology) tominutes.

The polynucleotides can be attached, via conventional technology, to asolid support in the form of a microarray, also known as a “DNA chip,” a“DNA microarray,” a “gene array”, a “gene chip,” and a “genome chip,” orin the form of a macroarray. In this context, an array is an orderlyarrangement of samples that enables the matching of known and unknownDNA samples, founded on base-pairing rules, and the automation ofidentifying the unknowns.

“Microarray” and “macroarray” are relative terms, distinguished fromeach other in terms of the diameter of the sample spots involved. Thus,microarray sample spots typically are about 200 microns in diameter orless, making it possible to array thousands of sample spots on a singlechip. These arrays can be prepared by hand but, preferably, are madeusing specialized robotics and read by means of specialized imagingequipment, particularly with spots with diameters in the lower-endrange. By contrast, macroarray sample spots typically are greater than200 microns in diameter, making them suitable for imaging by gel andblot scanners. Macroarrays may be prepared by hand using standardmicroplates or standard blotting membranes.

A microarray containing immobilized polynucleotides can be prepared by anumber of well-known approaches including, for example, light-directedmethods, such as VLSIPS™ (see U.S. Pat. No. 5,143,854), mechanicalmethods such as described in PCT No. 92/10183 or U.S. Pat. No.5,384,261, bead-based methods described, for example, in PCTUS/93/04145, and pin-based methods as detailed in U.S. Pat. No.5,288,514, inter alia. U.S. Pat. No. 5,556,752 to Lockhart describes thepreparation of a library of different, double-stranded polynucleotidesas a microarray, using the VLSIPS,™ and this approach also is suitablefor preparing a library of polynucleotides in a microarray. Flow channeltechniques, as described U.S. Pat. No. 5,677,195 and No. 5,384,261, alsocan be employed to prepare a microarray chip that has a variety ofdifferent immobilized polynucleotides. In this case, certain activatedregions of the substrate are mechanically separated from other regionswhen the polynucleotides are delivered through a flow channel to thesupport. As noted, the Lockhart '752 patent describes flow channelmethodology in some detail, including the use of protectivecoating-wetting facilitators to enhance the directed channeling ofliquids though designated flow paths.

Spotting methods also can be used to prepare a microarray biochip with avariety of immobilized polynucleotides. In this case, reactants aredelivered by directly depositing relatively small quantities in selectedregions of the support. In some steps, of course, the entire supportsurface can be sprayed or otherwise coated with a particular solution.In particular formats, a dispenser moves from region to region,depositing only as much polynucleotides or other reagent as necessary ateach stop. Typical dispensers include a micropipette, nanopippette,ink-jet type cartridge or pin to deliver the polynucleotide containingsolution or other fluid to the support and, optionally, a robotic systemto control the position of these delivery devices with respect to thesupport. In other formats, the dispenser includes a series of tubes ormultiple well trays, a manifold, and an array of delivery devices sothat various reagents can be delivered to the reaction regionssimultaneously.

Spotting methods are well known and include, for example, thosedescribed in U.S. Pat. No. 5,288,514, No. 5,312,233 and No. 6,024,138.In some cases, a combination of flowing channel and “spotting” onpredefined regions of the support also can be used to prepare microarraybiochips with immobilized polynucleotides.

Information and examples of constructing microarrays are readilyavailable in the art, such as in Cheung et al., Nature Genetics, Vol. 21(suppl.), pp. 15-19 (1999); Lipshutz et al., Nature Genetics, Vol. 21(suppl.), pp. 20-24 (1999); Brown & Botstein, Nature Genetics, Vol. 21(suppl.), pp. 33-37 (1999); Shoemaker et al., Nature, Vol. 409, pp.922-27 (2001); Schena et al., Science (1995) 467-470; Schena et al.,P.N.A.S. U.S.A. (1996) 93: 10614-10616; Pietu et al., Genome Res. (June1996) 6: 492-503; Zhao et al., Gene (Apr. 24, 1995) 156: 207-213;Soares, Curr. Opin. Biotechnol. (October 1997) 8: 542-546; Raval, J.Pharmacol Toxicol Methods (November 1994) 32: 125-127; Chalifour et al.,Anal. Biochem (Feb. 1, 1994) 216: 299-304; Stolz & Tuan, Mol.Biotechnol. (December 1996) 6: 225-230; Hong et al., Bioscience Reports(1982) 2: 907; and McGraw, Anal. Biochem. (1984) 143: 298.. Numerouspatents related to microarray development include U.S. Pat. No.5,082,830, No. 6,110,426, No. 6,004,755, No. 5,445,934, No. 5,532,128,No. 5,556,752, No. 5,242,974, No. 5,384,261, No. 5,405,783, No.5,412,087, No. 5,424,186, No. 5,429,807, No. 5,436,327, No. 5,472,672,No. 5,527,681, No. 5,529,756, No. 5,545,531, No. 5,554,501, No.5,561,071, No. 5,571,639, No. 5,593,839, No. 5,599,695, No. 5,624,711,No. 5,658,734 and No. 5,700,637, and PCT application WO 97/27317.

C. Hybridization and Analysis

Hybridization conditions (either initial hybridization without washingor a combination of initial hybridization and washing) can be selectedthat allow the DNA fragments to hybridize the complementary immobilizedpolynucleotides of the solid support and avoid unwanted crosshybridization. See e.g., Sambrook (1989), Chapter 9.47-9.62. In general,DNA annealing is carried out in high ionic strength to maximize theannealing rate and at temperatures about 20-25° C. below the meltingtemperature of the DNA (T_(m)). Various agents may be used to reducenon-specific binding (e.g. Denhardt's solution, BLOTTO, heparin, anddenatured salmon sperm DNA). Washing conditions should be as stringentas possible, generally sufficient salt and at a temperature 12-20° C.below the T_(m). Washing conditions may be determined empirically inpreliminary experiments. Other approaches may be used and may readily bedetermined by one of ordinary skill in the art.

The amount of amplified DNA that hybridizes to the solid support iscompared to a control to determine whether the extent of methylation ofone or more regions of DNA in a test sample is different from thecontrol sample. In one embodiment, the control can be an actual sampleof DNA such as genomic DNA from a normal healthy tissue that isprocessed in parallel with the test sample. Such control sample may beobtained from the same type of cell, tissue or organ as that from whichthe test sample was obtained. Alternatively, a control sample maycontain one or more selected DNA fragments, which may be prepared sothat it is fully methylated, fully unmethylated, or partially methylatedas desired. The control also can be a theoretical control whichrepresents a historical value derived from prior experimentation.

In a preferred embodiment, test and control DNA fragments are separatelylabeled with Cy-3 or Cy-5 dye. The labeled test and control fragmentsare then mixed together and hybridized to the solid support. Afterwashing, the slide is scanned with an array scanner either successivelyor simultaneously at the excitation wavelength of 635 nm (Cy5) and 532nm (Cy3). The images for each wavelength are recorded, pseudo-colors areapplied to each image (red for Cy5 and green for Cy3) and the images areoverlaid to show the relative density differences. The densities at bothwavelength for each spot are also quantified and analyzed. The extent ofmethylation (“methylation status”) of DNA in a test sample relative to acontrol sample is then determined either visually by the color of eachspot or through the measured density for each scanned channel.

Due to the qualitative nature of the present method achieved through thecompetitive amplification and hybridization, a very high degree ofsensitivity, enabling the detection of subtle changes in methylationlevel for each region is attainable. This is highly desirable becausetissue samples often contain mixed cell populations.

Applications of the Method

The present invention provides a high throughput methodology fordetecting methylation changes at specific regions throughout the genome.High throughput can be accomplished using well known systems involvingrobotic control, automated liquid handling, automatic spotting, plateand slide loaders, pin arrays (96, 384, 1536 etc.), and the like (seee.g., automated array systems from Cartesian Technologies Inc.). Thiswill allow tens of thousands of such regions, both CpG islands andnon-island regions, to be tested simultaneously so that any methylationchange in the genome can be detected. Also, hundreds or more samplesfrom different individuals can be analyzed, and changes in methylationthat are linked with a particular disease can later be exploited as adiagnostic screen for the disease. Other applications for the presentinvention are detailed below.

Diagnosis: The information thereby gleaned from using the methods hereininforms diagnoses and prognoses that reflects, in whole or in part, thegene-silencing pattern characterizing a particular disease. That is, themethylation determined via the present invention offers a disease-stateindicator of exceptional selectivity, specificity, and sensitivity.Furthermore, aberrant CpG island-hypermethylation, which occurs at highfrequency in tumors, can yield diagnostic information as well.Determining patient's genome methylation by means of the presentinvention opens the way, in a cost-effective manner, for anunprecedented early warning diagnosis. Inappropriate methylation changesin CpG islands is one of the earliest known markers in the developmentof many cancers and direct detection of these molecular aberrationsusing the methods disclosed herein provides an extraordinary opportunityfor unprecedented early stage molecular diagnosis of cancer. Inaddition, epigenetic changes have been implicated in several otherimportant diseases. These include atherosclerosis (Post et al., 1999),Angelman syndrome (Lalande et al., 1999), Duchenne muscular dystrophy(Yoshioka et al., 1998) and ICF syndrome (Kondo et al., 2000), to name afew.

Enabling technology for improved clinical trials: The methods disclosedherein can be used to determine which individuals are afflicted withmethylation dependent cancers. This can increase the success of efficacystudies in clinical trials of drugs targeting the basis for themethylation difference in the cancer. Drug candidates that effectmethylation status represent the next generation of non-cytotoxic cancertherapies. In addition, since methylation status of a gene oftencorrelates with its expression, methylation profile of test subjects canalso be used in patient selection in clinical trials of other drugs,including cancer drugs.

Personalized Medicine: With the high cost of cancer therapies and thewide variety of cancer types, it is important that tests be developed todetermine which patients will respond to which therapies. As the newgeneration of methylation dependent cancer therapies advances, theassays of the present invention will be important to determine whichcancer patients are afflicted with methylation defects. Such data canhelp the oncologist's therapy decision process, determining patientsuitability for a methylation-based drug regimen.

Discovery: Detection of inappropriate methylation in CpG islands acts asan indicator of which genes are involved in the development of cancer.Furthermore, only a small fraction (<3%) of all CpG islands have beeninvestigated as to their role in cancer. Discoveries of new genesilencing events in cancer using the methods described herein willprovide critical information for the initiation of new drug discoveryand optimization strategies.

Toxicology: The cost of bringing drug candidates through clinical trialsthat eventually fail due to toxicological problems is enormous. Thus,there is a great need for methylation detection methods to “weed-out”drugs with toxicology problems at an early (pre-clinical) stage.Applying the methods of the present invention in a high throughputscreening format will be helpful in determining if a particular drugimpacts the methylation status of cells or tissues. Such screening wouldlower the likelihood that candidate drugs with mutagentic orepigenetic-based toxicity will proceed inappropriately to clinicaltrials.

EXAMPLES Example 1 Direct Microarray Screening of DifferentiallyMethylated Sites (“DMS²) for a Mouse Transgene

This example demonstrates the use of an embodiment of the presentinvention for detecting the extent of methylation of a transgene inmouse DNA. The method involves a DNA amplification step and is referredto as DMS² (direct microarray-based screening of differentiallymethylated sites).

Oligonucleotides of 50-60 nucleotides in length were synthesized forhybridizing to selected CpG islands in mouse genes and for hybridizingto a mouse transgene known as HRD (Engler et al., (1991) Cell65(6):939-47). HRD provides a useful control DNA in the assay becausethe transgene is highly methylated in one mouse strain (C57BL/6J,abbreviated as B) and unmethylated in another (DBA/2J, abbreviated asD), and HRD is integrated in the mouse genome in amounts comparable withother mouse genes. FIG. 5A is a Southern blot that demonstrates that thetransgene is methylated in strain B but not in strain D.

DNA (1 μg) from each HRD transgenic mouse strain was separately digestedwith five units of HpaII in 50 μl for 6 hours at 37° C. The digestionmixture was heat inactivated at 65° C. for 15 minutes, precipitated withethanol, and resuspended in a volume of 20.5 μl. For ligation, twomicroliters of 100 μM of oligonucleotide-linker, 2.3 μl of 10× ligationbuffer and 0.2 μl T4 DNA ligase (both from New England Biolabs, Beverly,Mass.) were added to the samples and the mixture incubated at roomtemperature for 6-16 hours. The oligonucleotide-linker is shown in FIG.4A.

The ligated DNA fragments were purified using a Qiagen MinElute PCRpurification Kit based on silica gel-membrane spin columns (Qiagen,Valencia, Calif.). Samples were processed in accordance with themanufacturer's instructions except that a 30% guanidine hydrochloridewash step was added prior to the regular wash. The product was eluted in25 μl (15 μl+10 μl) elution buffer provided in the kit.

For PCR, 2.5 μl of the eluted DNA was amplified with AmpliTaq Gold® in50 μl reaction with Gold buffer (Applied Biosystems, Foster City,Calif.), 100 μM dNTP, 1.2 M betaine, 1 μM Cyanine dye-labeledamplification primer (Cy-3 for the methylated mouse strain DNA and Cy-5for the unmethylated mouse strain DNA) and 1 Unit of DNA polymerase. Theprimer sequence was as follows: Dye-5′-CTGACGATGAGTCCTGAGTCGG-3′ (SEQ IDNO:6)The cycle condition was 95° C. for 6.5 minutes followed by 35 cycles of94° C. for 30 sec, 55° C. for 20 seconds, and 72° C. for 1 minute. Theamplified products (i.e., amplicons) were purified with the Qiagen PCRPurification kit and eluted in 10 μl of elution buffer.

A second PCR reaction using transgene primers, followed by agarose gelelectrophoresis, was performed to determine if the amplicon containedthe expected fragment predicted by the assay design. The result in FIG.5B shows the expected strong amplification of transgene DNA fragment(see arrow) from the processed DNA of the unmethylated transgene mousestrain (lane D) while no amplification of a transgene DNA fragment wasseen from the processed DNA of the methylated transgene mouse strain(lane B). The presence of a Cy-3 or Cy-5 label on the DNA amplicon didnot affect the results.

Microarrays were prepared by spotting polynucleotides in triplicate ontoa glass slide using a mechanical arrayer (Affymetrix, Santa Clara,Calif.). The Cy-3 and Cy-5 labeled DNA amplicons were mixed together andhybridized to the array in 6×SSC at 55° C. for several hours. Thewashing conditions were 2×SSC and 0.03% SDS; 2×SSC; 1×SSC; 0.2×SSC, allat room temperature. Arrays were scanned at 635 nm for the Cy-5 and 532nm for Cy-3.

As shown in FIG. 5C, the oligonucleotide probe for the transgenedetected higher Cy5 (from strain D) hybridization signal than Cy3 (fromstrain B) signal consistently in the triplet, indicating the successfuldetection of the methylation difference for the transgene. The controlprobe, as expected, detected a similar level of hybridization signal inboth Cy5 and Cy3 channels consistent with the lack of methylation in thecontrol gene in the two strains.

Example 2 Screening of Differentially Methylated Sites Using a cDNAMicroarray

This example demonstrates the use of an embodiment of the presentinvention for detecting the extent of methylation of mouse genes using ahigh-density cDNA microarray. The mixture of Cy-3 and Cy-5 labeled DNAamplicons were prepared as described in Example 1 and hybridized to theM9K mouse cDNA microarray, which contains approximately 9000sequence-verified cDNA clones spotted on a microscope slide. Thehybridized array was scanned at 532nm and 635 nm as described in Example1.

An overlay of the two scans was achieved by pseudo-coloring (green forCy-3 and red for Cy-5). Four independent hybridizations were performedand the results indicate a high degree of reproducibility. Most of thespots show a yellow color of weak intensity, indicating that the genomicregions for the clones are not differentially methylated (they havesimilar methylation level) and likely are unmethylated in both samples.Red or green spots were observed for about 50 clones which have aCy5/Cy3 ratio larger than 3 or smaller than 0.3, indicating a differentlevel of methylation in the genomic regions they represent. Many spotsshow very weak or no signal, indicating that the regions detected by theimmobilized cDNA are either highly methylated in both samples or are notrepresented in the amplicon (the latter may be due to the fact that thecDNA microarray contained many clones from the 3′ part of the genes thatmay not have CpG dinucleotides).

Example 3 Screening of Differentially Methylated Sites in Human BreastCancers Using a 762-Feature CpG Island Fragment Microarray

In this example, 2 μg of genomic DNA from three human breast tumor celllines, T47D, MB435, and SKBR3, were digested with HpaII overnight at 37°C. One fourth of the digested DNA was ligated to 10 μl of 100 μM of theunphosphorylated linker shown in FIG. 2 in 30 μl reaction at 16° C. for5-16 hours. A 0.2 ml PCR tube contained the following mixture: 10 μl ofthe ligation product, 10 μl of 10× reaction buffer, 10 μl of 2 mM dNTPs,20 μl of 5M betaine (Sigma-Aldrich, St Louis, Mo.), 5 μl DMSO, 44.5 μlH2O, and 0.5 μl (2.5 units) of Taq polymerase (Applied Biosystems,Foster City, Calif.). PCR conditions were as follows: 72° C., 5 minutes,then 94° C., 3 minutes followed by 25 cycles of 94° C., 30 seconds; 60°C., 30 seconds; 72° C., 3 minutes. The amplified samples were stored at4° C. Amplified products (amplicons) were purified with Qiagen PCRpurification kit (Qiagen, Valencia, Calif.). 2 μg of each amplicon waslabeled either with Cy3 (for T47D) or Cy5 (for MB435 and SKBR3) dCTP togenerate labeled probes as follows: 2 μg of amplicon (diluted in 21 μlwith H₂O) and 20 μl of 2.5× random primer/reaction buffer (125 mM TrispH 6.8, 12.5 mM MgCl2, 25 mM 2-mercaptoethanol, 750 μg/ml randomoctamers) are combined in a 0.5 ml eppendorf tube, incubated in boilingwater for 5 minutes, placed on ice for 5 minutes. The samples werecentrifuged briefly and the liquid fraction placed on ice, and thefollowing was added: 5 μl 10× dNTP mix (1.2 mM each of dATP, dGTP anddTTP, 0.6 mM dCTP, 10 mM Tris pH8.0, 1 mM EDTA); 3 μl Cy5 or Cy3 dCTP(Amersham Pharmacia, 1 mM), and 1 μl klenow fragment (New EnglandBiolabs). The mixture was incubated at 37° C. for 1.5 hours. The labeledprobes were purified with a Microcon 30 filter (Amicon/Millipore).

The purified probes were mixed to form two mixtures: T47D/Cy3 withMB435/Cy5 and T47D/Cy3 with SKBR3/Cy5. The microarray composing of 762CpG island fragments was manufactured as described in Example 1. Thedye-labeled mixtures were hybridized to the microarray in a 65° C. waterbath for 16 hours and washed and scanned as described (Example 1).

The results showed 25 CpG island fragments with significant differentialhybridization signals (a ratio of Cy5/Cy3 greater than 3 or smaller than0.3) indicating differential methylation between the cell lines.Bisulfite-based quantitative methylation assay COBRA (Xiong & Laird,Nucleic Acids Res. Jun. 15, 1997;25(12):2532-2534) was used to verifythe detected differences in methylation among the samples. The COBRAassays confirmed differential methylation for six of nine tested CpGislands fragments.

The invention thus has been disclosed broadly and illustrated inreference to representative embodiments described above. Those skilledin the art will recognize that various modifications can be made to thepresent invention without departing from the spirit and scope thereof.

1. A method for detecting whether the extent of methylation of one ormore regions of DNA in a test sample is different from that of acontrol, comprising: generating DNA fragments from al least one testsample of DNA by cleaving methylation sites in the DNA that are notinethylatped while sparing methylation sites in the DNA that aremethylated; ligating an oligonucleotide-linker to the ends; of the DNAfragments; amplifying the DNA fragments by initiating DNA amplificationat the oligonucleotide linker with a linker-primer; hybridizing theamplified DNA to one or more polynucleotides immobilized on a solidsupport, said polynucleotides being complementary to one or more regionsof DNA in the test sample; and comparing the amount of amplified DNAfrom the test sample that hybridizes to the immobilized polynucleotidesversus that of a control, thereby detecting whether the extent ofmethylation of the one or more regions of DNA is different between thetest sample and the control.
 2. The method of claim 1, wherein saidcontrol is one or more control samples of DNA processed the same as theone or more test samples of DNA.
 3. The method of claim 1, wherein saidDNA fragments are generated by cleaving with a methylation-sensitiveagent.
 4. The method of claim 3, wherein said methylation-sensitiveagent is a methylation sensitive restriction enzyme.
 5. The method ofclaim 4, wherein said restriction enzyme cleaves a recognition sequenceselected from the group consisting of CCGG, CCGC, GCGC, ACGT, CGGCCG,GCCGGC, GGCGCC, CCCGGG, CGCG, ATCGTA, TTCGAA, GTCGAG, and CTCGAG.
 6. Themethod of claim 4, wherein said methylation-sensitive restriction enzymecleaves a recognition sequence selected from the group consisting of:CGGCCG, GCCGGC, GGCGCC, CCCGGG, CCGG, CCGC, GCGC, and ACGT.
 7. Themethod of claim 1 wherein said oligonucleotide linker is phosphorylated.8. The method of claim 1 wherein said oligonucleotide linker is notphosphorylated.
 9. The method of claim 8 wherein said non phosphorylatedoligonucleotide linker is double stranded and wherein only one strand ofthe linker is ligated to the fragment.
 10. The method of claim 9 whereina polymerase chain mediated extension of the ligated fragment precedesthe step of amplification and wherein one chain of the oligonucleotidelinker is the linker primer.
 11. The method of claim 1, furthercomprising the step of cleaving the DNA with at least onemethylation-insensitive agent.
 12. The method of claim 11, wherein saidmethylation-insensitive agent is a methylation-insensitive restrictionenzyme.
 13. The method of claim 12, wherein said methylation insensitiverestriction enzyme is selected from the group consisting of EcoRI, ApoI,Tsp509I, MseI, BfaI, Csp6I, NlaIII, DpnII, and CviJI.
 14. The method ofclaim 11, wherein said cleavage by the methylation-sensitive agent andthe methylation-insensitive agent occurs simultaneously in the samemixture.
 15. The method of claim 11, further comprising the step ofligating a non-phosphorylated oligonucleotide-linker to the end of eachDNA fragment generated by the at least one methylation-insensitiveagent.
 16. The method of claim 15, wherein said oligonucleotide-linkersthat ligate to sites cleaved by the methylation-insensitive agent aredistinct from the oligonucleotide-linkers that ligate to the sitescleaved by the methylation-sensitive agent.
 17. The method of claim 1,wherein said oligonucleotide-linker is a universaloligonucleotide-linker.
 18. The method of claim 1, wherein said step ofcomparing the amount of amplified DNA that hybridizes is performed bymeasuring a detectable moiety associated with the amplified DNA.
 19. Themethod of claim 18, wherein said detectable moiety is a cyanine dye. 20.The method of claims 18, wherein said detectable moiety is a labeleddendrimer.
 21. The method of claim 18, wherein said detectable moiety isassociated with the amplification primer prior to its incorporation intoamplified product.
 22. The method of claims 18, wherein said detectablemoiety is attached to the amplified DNA following amplification.
 23. Themethod of claim 18, wherein said detectable moiety is incorporated intothe synthesized DNA during amplification.
 24. The method of claim 1,wherein said immobilized polynucleotides comprise sequencescomplementary to one or more CpG islands.
 25. The method of claim 1,wherein said immobilized polynucleotides comprise sequencescomplementary to one or more gene regulatory sequences or an encodinggene sequence.
 26. The method of claim 1, wherein said immobilizedpolynucleotides comprise at least two polynucleotides immobilized as anarray.
 27. The method of claim 26, wherein said array is a microarray.28. The method of claim 1, wherein said step of amplifying DNA occursvia the polymerase chain reaction.
 29. The method of claim 2, whereinsaid DNA fragments from the test and control samples are labeled withdifferent detectable moieties that are distinguishable from each otherwhen mixed together.
 30. The method of claim 29, wherein said differentdetectable moieties comprise a cyanine-3 dye and a cyanine-5 dye. 31.The method of claim 29, wherein said test and control samples are mixedtogether before the step of hybridization.
 32. A method for detectingwhether the extent of methylation of one or more regions of DNA in atest sample is different from that of a control, comprising: generatingDNA fragments from at least one test sample of DNA by cleavingmethylation sites in the DNA that are not methylated while sparingmethylation sites in the DNA that are methylated; ligating anoligonucleotide-linker to the ends of the DNA fragments; selecting aparticular size range of the DNA fragments; labeling the DNA fragmentswith a detectable moiety; hybridizing the DNA fragments to one or morepolynucleotides immobilized on a solid support, said polynucleotidesbeing complementary to one or more regions of DNA in the test sample;and comparing the amount of the detectable moiety associated with theimmobilized polynucleotides for the test sample versus a control,thereby detecting whether the extent of methylation of the one or moreregions of DNA is different between the test sample and the control. 33.The method of claim 32, wherein said control is one or more controlsamples of DNA processed the same as the one or more test samples ofDNA.
 34. The method of claim 32, wherein said DNA fragments aregenerated by cleaving with a methylation-sensitive agent.
 35. The methodof claim 34, wherein said methylation-sensitive agent is a methylationsensitive restriction enzyme.
 36. The method of claim 35, wherein saidrestriction enzyme cleaves a recognition sequence selected from thegroup consisting of CCGG, CCGC, GCGC, ACGT, CGGCCG, GCCGGC, GGCGCC,CCCGGG, CGCG, ATCGTA, TTCGAA, GTCGAG, and CTCGAG.
 37. The method ofclaim 35, wherein said methylation-sensitive restriction enzyme cleavesa recognition sequence selected from the group consisting of: CGGCCG,GCCGGC, GGCGCC, CCCGGG, CCGG, CCGC, GCGC, and ACGT.
 38. The method ofclaims 32, wherein said detectable moiety is a labeled dendrimer. 39.The method of claim 32, wherein said one or more immobilizedpolynucleotides comprise sequences complementary to a one or more CpGislands.
 40. The method of claim 32, wherein said one or moreimmobilized polynucleotides comprise sequences complementary to one ormore gene regulatory sequences or an encoding gene sequence.
 41. Themethod of claim 32, wherein said one or more immobilized polynucleotidescomprise at least two polynucleotides immobilized as an array.
 42. Themethod of claim 41, wherein said array is a microarray.
 43. The methodof claim 33, wherein said DNA fragments from the test and controlsamples are labeled with different detectable moieties that aredistinguishable from each other when mixed together.
 44. The method ofclaim 43, wherein said different detectable moieties comprise acyanine-3 dye and a cyanine-5 dye.
 45. The method of claim 43, whereinsaid test and control samples are mixed together before the step ofhybridization.
 46. The method of claim 32, wherein said DNA is labeledby labeling the oligonucleotide-linker.
 47. The method of claim 46,wherein said oligonucleotide-linker comprises a capture sequence usedfor attaching the detectable label.
 48. The method of claim 32, whereinsaid step of selecting a particular size range of the DNA fragments isconducted after the step of linker ligation.
 49. The method of claim 32,wherein said step of selecting a particular size range of the DNAfragments step results in the separation of fragments about 1,500 basepairs in length or less.
 50. A method of detecting whether an individualhas a disease state associated with an abnormal extent of methylation ofone or more regions of DNA in the individual, said method comprising:providing a test sample of DNA derived from tissue of the individual;determining the extent of methylation for one or more regions of DNA inthe sample from the individual in accordance with the method of claim 1;and determining abnormal methylation by relating the extent ofmethylation of the one or more regions of DNA in the test sample to thatof a normal healthy control, thereby indicating if the individual hasthe state of disease.
 51. A method of detecting whether an individualhas a disease state associated with an abnormal extent of methylation ofone or more regions of DNA in the individual, said method comprising:providing a test sample of DNA derived from tissue of the individual;determining the extent of methylation for one or more regions of DNA inthe DNA of sample from the individual in accordance with the method ofclaim 32; and determining abnormal methylation by relating the extent ofmethylation of the one or more regions of DNA in the test sample to thatof a normal healthy control, thereby indicating if the individual hasthe state of disease.
 52. A method of establishing whether the extent ofmethylation in a DNA region of an individual correlates with a diseasestate, said method comprising: determining the extent of methylation forsaid region in the DNA for various individuals with the disease stateand individuals without the disease state in accordance with the methodof claim 1 and then determining if the extent of methylation of theregion correlates with the disease state.
 53. A method of establishingwhether the extent of methylation in a DNA region of an individualcorrelates with a disease state, said method comprising: determining theextent of methylation for said region in the DNA for various individualswith the disease state and individuals without the disease state inaccordance with the method of claim 32 and then determining if theextent of methylation of the region correlates with the disease state.